Disease model animal carrying foreign ppar alpha gene transferred thereinto and use thereof

ABSTRACT

The present invention provides a non-human mammal, or a part of its living body, which stably retains a DNA encoding a heterologous PPARα in an expressible state, and has one or more different genetic modifications resulting in a pathological condition identical or similar to a disease associated with the regulation of PPARα activity or a foreign DNA under the control of a promoter having PPRE, as well as a method of screening for agonists/antagonists for the heterologous PPARα using the animal.

TECHNICAL FIELD

The present invention relates to a heterologous PPARα gene-introducednon-human mammal. To be specific, the present invention relates to anon-human mammal into which a heterologous PPARα gene is introduced andwhich has a genetic modification showing a phenotype usable as anexperimental animal model for a disease associated with the regulationof PPARα activity, a method of screening for an agent for theprophylaxis/treatment of various diseases associated with the regulationof PPARα activity, using the same, in the mammal from which theheterologous PPARα has been derived, and an agent for theprophylaxis/treatment of said diseases obtainable by said method.

BACKGROUND ART

Peroxisome Proliferator-Activated Receptor (hereinafter abbreviated asPPAR) α is a nuclear receptor type transcription factor that is highlyexpressed in liver, heart, kidney and the like, and plays a central rolein lipid homeostasis through controlling the expression of genesencoding various proteins associated with lipid metabolism such asenzymes associated with β-oxidation of fatty acids and apolipoproteins.PPARα forms a heterodimer with retinoid X receptor (RXR), and binds, inthe presence of its ligand, to Peroxisome Proliferator-ResponsiveElement (PPRE) sequence located in the 5′ upstream of a target gene topromote the transcription of the gene. On the other had, it is knownthat PPARα also suppresses the expression of some genes in the presenceof its ligand. It is thought to be due to the competition for thebinding to a target DNA sequence or co-activator with othertranscription factors.

PPARα is activated by a fat-soluble molecule such as a long-chainunsaturated fatty acid and a drug of fibrate class. A PPARα agonistenhances fatty acid-oxidation activities in liver, on the other hand, itsuppresses the expression of apolipoprotein C-III (apoC-III; a mainconstituent of plasma lipoproteins together with neutral fats (TG)). Itis therefore effective to reduce the blood TG concentration. Since itpromotes the expression of apolipoprotein A-I (apoA-I) andapolipoprotein A-II (apoA-II), which are main constituents of highdensity lipoprotein-cholesterols (HDL-C), it has a blood HDL-Cconcentration-increasing effect. Therefore, a PPARα agonist is actuallyused as an agent for the prophylaxis/treatment of hyperlipidemiaincluding hypertriglyceridemia and hypo-HDL-cholesterolemia, andarteriosclerosis (for example, refer to Jean-Charles Fruchart et al.,Current Atherosclerosis Reports, (USA), 2001, Vol. 3 (No. 1), p. 83-92).A PPARα agonist is also known to show a heart-protective effect againstischemia (for example, refer to Antonia Tabernero et al., BMCPharmacology, (UK), Apr. 9, 2002 (published on-line), BioMed Central,internet <http://www.biomedcentral.com/1471-2210/2/10>).

A development of a novel PPARα agonist typically proceeds in thefollowing order:

carrying out in vitro screening such as a binding assay using humanPPARα or expression assay of a gene under the control of PPRE (e.g.,apoA-I, PPRE-reporter chimeric gene, etc.) in a human cell line, and

evaluating in vivo effect of the compounds that has been confirmed tohave a high activity by administration to an experimental animal such asmouse, rat, or the like. However, due to the differences in PPARαstructure between human and other mammals (for example, the amino acididentity between human and mouse is 92%), some drugs may show anagonistic activity only against human PPARα. Since such drugs show noactivity against the endogenous PPARα of the experimental animal, theycannot be evaluated using existing animal models.

It is therefore an object of the present invention to provide a novelanimal model allowing effective evaluation of the in vivo effect of aPPARα agonist showing an agonistic activity only against human PPARα,and a method of screening for an agent for the prophylaxis/treatment ofvarious diseases such as hyperlipidemia, combined dyslipidemia,arteriosclerosis, hypertension, thrombosis, ischemic heart diseases, andischemic brain diseases using said animal model.

DISCLOSURE OF THE INVENTION

The present inventors conducted extensive investigations with the aim ofaccomplishing the above-described object, and produced a transgenicmouse which stably retains a DNA encoding human PPARα in an expressiblestate. When a compound that has an activity against human PPARα but hasno activity against mouse PPARα was administered to the mouse, it hasbeen shown that said mouse showed various characteristic responsesinduced by a peroxisome proliferator including peroxisome proliferationand enhancement of the expression of lipid metabolism-related genes.Furthermore, the present inventors succeeded in producing human PPARαexpressing animal models for various diseases associated with thecontrol of PPARα activity including hyperlipidemia and arteriosclerosisby crossing the obtained human PPARα expressing transgenic mouse andexperimental animal models for said diseases.

The present inventors conducted further investigations based on thesefindings, which resulted in the completion of the present invention.

Accordingly, the present invention provides:

[1] a non-human mammal, which stably retains a DNA encoding aheterologous PPARα in an expressible state and has one or more differentgenetic modifications, or a part of its living body;

[2] the animal or the part of its living body of the above-mentioned[1], wherein at least one of said different genetic modificationsresults in pathological condition(s) equal or similar to disease(s)associated with the regulation of PPARα activity;

[3] the animal or the part of its living body of the above-mentioned[1], wherein at least one of said different genetic modifications isintroduction of a foreign DNA under the control of a promoter havingPPRE;

[4] the animal or the part of its living body of the above-mentioned[1], wherein said heterologous PPARα is human derived PPARα;

[5] the animal or the part of its living body of the above-mentioned[1], wherein said heterologous PPARα has the same or substantially thesame amino acid sequence as the amino acid sequence represented by SEQID NO: 2;

[6] the animal or the part of its living body of the above-mentioned[1], wherein said non-human mammal is rabbit, dog, cat, guinea pig,hamster, mouse or rat;

[7] the animal or the part of its living body of the above-mentioned[1], wherein said non-human mammal is mouse;

[8] the animal or the part of its living body of the above-mentioned[1], wherein said animal expresses said heterologous PPARα in place oflacking its endogenous PPARα;

[9] the animal or the part of its living body of the above-mentioned[8], wherein said animal is obtainable by crossing an endogenousPPARα-deficient animal and the same species of animal that expresses aheterologous PPARα;

[10] the animal or the part of its living body of the above-mentioned[8], wherein said endogenous PPARα is mouse-derived PPARα and saidheterologous PPARα is human-derived PPARα;

[11] the animal or the part of its living body of the above-mentioned[2], wherein said diseases associated with the regulation of PPARαactivity are one or more diseases selected from the group consisting ofhyperlipidemia, hypertriglyceridemia, combined dyslipidemia,hypo-HDL-cholesterolemia, arteriosclerosis, peripheral arterialobstruction, intermittent claudication, gangrene, hypertension,thrombosis, ischemic heart disease, acute myocardial infarction, heartfailure, congestive heart failure, unstable angina pectoris, post-PTCArestenosis, post-stenting restenosis, hyperfibrinogemia, cardiomyopathy,cerebral hemorrhage, transient ischemic attack, cerebral infarction,cerebral apoplexy, chronic glomerulonephritis, diabetic nephropathy,renal arteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia,gonadal dysfunction, liver cancer, breast cancer and endometritis;

[12] the animal or the part of its living body of the above-mentioned[1], wherein said heterologous PPARα is specifically expressed in one ormore region selected from the group consisting of liver, heart, kidney,adrenal gland, blood vessel, gastrointestinal tract and brain;

[13] the animal or the part of its living body of the above-mentioned[1], wherein said heterologous PPARα is specifically expressed in liver;

[14] a method of screening for an agonist or antagonist for aheterologous PPARα, which comprises applying a test substance to theanimal or the part of its living body of the above-mentioned [1], andassaying its agonistic or antagonistic activity against the heterologousPPARα;

[15] a method of screening for an agonist or antagonist for aheterologous PPARα, which comprises applying a test substance to theanimal or the part of its living body of the above-mentioned [3], andassaying its agonistic or antagonistic activity against the heterologousPPARα using the expression of a foreign DNA under the control of apromoter having PPRE as an index; and

[16] a method of screening for a substance having aprophylactic/therapeutic activity for disease(s) associated with theregulation of PPARα activity in an animal from which a heterologousPPARα is derived, which comprises administering a test substance to theanimal of the above-mentioned [2], and assaying effect(s) of thesubstance on pathological condition(s) equal or similar to disease(s)associated with the regulation of PPARα activity in the animal.

Furthermore, the present invention provides:

[17] an agonist for a heterologous PPARα obtainable by the method of theabove-mentioned [14] or [15];

[18] an agent for the prophylaxis/treatment of one or more diseasesselected from the group consisting of hyperlipidemia,hypertriglyceridemia, combined dyslipidemia, hypo-HDL-cholesterolemia,arteriosclerosis, peripheral arterial obstruction, intermittentclaudication, gangrene, hypertension, thrombosis, ischemic heartdisease, acute myocardial infarction, heart failure, congestive heartfailure, unstable angina pectoris, post-PTCA restenosis, post-stentrestenosis, hyperfibrinogemia, cardiomyopathy, cerebral hemorrhage,transient ischemic attack, cerebral infarction, cerebral apoplexy,chronic glomerulonephritis, diabetic nephropathy, renalarteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia andgonadal dysfunction in an animal from which a heterologous PPARα isderived, which comprises the agonist of the above-mentioned [17];

[19] the agent for the prophylaxis/treatment of the above-mentioned[18], wherein said animal from which the heterologous PPARα is derivedis human;

[20] a method for the prophylaxis/treatment of one or more diseasesselected from the group consisting of hyperlipidemia,hypertriglyceridemia, combined dyslipidemia, hypo-HDL-cholesterolemia,arteriosclerosis, peripheral arterial obstruction, intermittentclaudication, gangrene, hypertension, thrombosis, ischemic heartdisease, acute myocardial infarction, heart failure, congestive heartfailure, unstable angina pectoris, post-PTCA restenosis, post-stentrestenosis, hyperfibrinogemia, cardiomyopathy, cerebral hemorrhage,transient ischemic attack, cerebral infarction, cerebral apoplexy,chronic glomerulonephritis, diabetic nephropathy, renalarteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia andgonadal dysfunction in an animal from which a heterologous PPARα isderived, which comprises administering to said animal an effectiveamount of the agonist of the above-mentioned [17];

[21] a use of the agonist of the above-mentioned [17] for the productionof an agent for the prophylaxis/treatment of one or more diseasesselected from the group consisting of hyperlipidemia,hypertriglyceridemia, combined dyslipidemia, hypo-HDL-cholesterolemia,arteriosclerosis, peripheral arterial obstruction, intermittentclaudication, gangrene, hypertension, thrombosis, ischemic heartdisease, acute myocardial infarction, heart failure, congestive heartfailure, unstable angina pectoris, post-PTCA restenosis, post-stentrestenosis, hyperfibrinogemia, cardiomyopathy, cerebral hemorrhage,transient ischemic attack, cerebral infarction, cerebral apoplexy,chronic glomerulonephritis, diabetic nephropathy, renalarteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia andgonadal dysfunction in an animal from which a heterologous PPARα isderived;

[22] an antagonist for a heterologous PPARα obtainable by the method ofthe above-mentioned [14] or [15];

[23] an agent for the prophylaxis/treatment of one or more diseasesselected from the group consisting of liver cancer, breast cancer andendometritis in an animal from which a heterologous PPARα is derived,which comprises the antagonist of the above-mentioned [22];

[24] the agent for the prophylaxis/treatment of the above-mentioned[23], wherein said animal from which the heterologous PPARα is derivedis human;

[25] the method for the prophylaxis/treatment of one or more diseasesselected from the group consisting of liver cancer, breast cancer andendometritis in an animal from which a heterologous PPARα is derived,which comprises administering to said animal an effective amount of theantagonist of the above-mentioned [22];

[26] a use of the antagonist of the above-mentioned [22] for theproduction of an agent for the prophylaxis/treatment of one or morediseases selected from the group consisting of liver cancer, breastcancer and endometritis in an animal from which a heterologous PPARα isderived;

[27] a substance having a prophylactic/therapeutic activity fordisease(s) associated with the control of PPARα activity in an animalfrom which a heterologous PPARα is derived, which is obtainable by themethod of the above-mentioned [16];

[28] an agent for the prophylaxis/treatment of disease(s) associatedwith the control of PPARα activity in an animal from which aheterologous PPARα is derived, which comprises the substance of theabove-mentioned [27];

[29] the agent for the prophylaxis/treatment of the above-mentioned[28], wherein said diseases associated with the control of theheterologous PPARα activity are one or more diseases selected from thegroup consisting of hyperlipidemia, hypertriglyceridemia, combineddyslipidemia, hypo-HDL-cholesterolemia, arteriosclerosis, peripheralarterial obstruction, intermittent claudication, gangrene, hypertension,thrombosis, ischemic heart disease, acute myocardial infarction, heartfailure, congestive heart failure, unstable angina pectoris, post-PTCArestenosis, post-stent restenosis, hyperfibrinogemia, cardiomyopathy,cerebral hemorrhage, transient ischemic attack, cerebral infarction,cerebral apoplexy, chronic glomerulonephritis, diabetic nephropathy,renal arteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia andgonadal dysfunction;

[30] a method for the prophylaxis/treatment of disease(s) associatedwith the control of PPARα activity in an animal from which aheterologous PPARα is derived, which comprises administering to saidanimal an effective amount of the substance of the above-mentioned [27];and

[31] a use of the substance of the above-mentioned [27] for theproduction of an agent for the prophylaxis/treatment of disease(s)associated with the control of PPARα activity in an animal from which aheterologous PPARα is derived, and the like.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a drawing showing a restriction enzyme map of a human PPARαexpression vector pKS-SEPP2. The arrow in the figure shows the directionof transcription.

BEST MODE FOR CARRYING OUT THE INVENTION

The heterologous PPARα gene-introduced non-human mammal of the presentinvention is an animal model allowing evaluation of the efficacy of aPPARα agonist or antagonist, in vivo, which acts on the heterologousPPARα, but not on the endogenous PPARα of the non-human mammal.

The non-human mammal stably retaining a DNA encoding a heterologousPPARα in an expressible state (hereinafter, also referred to as “thetransgenic non-human mammal of the present invention” or simply“transgenic animal of the present invention”) “stably retains” a DNAencoding a heterologous PPARα in an expressible state. “stably retains”means that the DNA encoding the heterologous PPARα durably exists in anexpressible state within the cells of said animal. Although said DNA maybe integrated into the host chromosome or stably exist as anextrachromosomal DNA, it is preferably retained in the state of beingintegrated into the host chromosome.

The transgenic animal of the present invention can be prepared bytransfecting a DNA encoding the heterologous PPARα of interest into afertilized egg, unfertilized egg, spermatozoon or precursor cell thereof(primordial germ cell, oogonium, oocyte, egg cell, spermatogonium,spermatocyte, sperm cell or the like), preferably in the early stage inthe embryogenic development (more preferably before the 8-cell phase) ofthe non-human mammal, by a gene transfer method, such as the calciumphosphate coprecipitation method, the electroporation method, thelipofection method, the agglutination method, the microinjection method,the particle gun method, the DEAE-dextran method, etc. Also, it ispossible to transfect the DNA of interest into a somatic cell, tissue,organ or the like of the non-human mammal by the gene transfer method,and utilize the transformant for cell culture, tissue culture, etc. Inaddition, these cells may be fused with the above-described embryo (orgerm) cell by a publicly known cell fusion method to produce thetransgenic animal. Alternatively, the transgenic animal can also beobtained, in the same manner as producing a knockout animal, bytransfecting the DNA of interest into embryonic stem cells (ES cells) ofthe non-human mammal using the above-described gene transfer method,selecting clones stably incorporating the DNA, injecting the ES cellsinto a blastocyst or aggregating an ES cell mass with 8-cell embryos toproduce chimeric mice, and selecting one in which the introduced DNA hasbeen transmitted to germ line.

A part of the living body of the transgenic animal produced in thismanner (for example, (1) a cell, tissue, organ or the like stablyretaining a DNA encoding a heterologous PPARα; (2) ones obtained byculturing and, if necessary, subculturing a cell or tissue derivedtherefrom, etc.) can be used for the same purpose as “the non-humanmammal stably retaining a DNA encoding a heterologous PPARα in anexpressible state” of the present invention, as “a part of the livingbody of the non-human mammal stably retaining a DNA encoding aheterologous PPARα in an expressible state” of the present invention.

As examples of the part of the living body of the transgenic animal ofthe present invention, organs such as the liver, heart, kidney, adrenalgland, blood vessels, gastrointestinal tract and brain, and tissuesections and cells derived from these organs, and the like can bementioned.

The “non-human mammal” that can be used as the subject of the presentinvention is not subject to limitation, as long as it is a non-humanmammal for which a transgenic system has been established; for example,bovine, monkey, swine, sheep, goat, rabbit, dog, cat, guinea pig,hamster, rat, mouse and the like can be mentioned. Preferably, rabbit,dog, cat, guinea pig, hamster, mouse, rat and the like can be mentioned;from the viewpoint of preparation of a disease model animal, inparticular, rodent animals, which have relatively short ontogeny andbiological cycles, and which permit easy propagation, are morepreferable, and mice (for example, C57BL/6 strain, DBA2 strain and thelike as pure strains, B6C3F₁ strain, BDF₁ strain, B6D2F₁ strain, BALB/cstrain, ICR strain and the like as crossed strains) and rats (forexample, Wistar, SD and the like) are particularly preferable.

Also, in addition to mammals, birds such as chicken can be used for thesame purpose as that of a “non-human mammal” that is the subject of thepresent invention.

“A DNA that encodes a heterologous PPARα” means a DNA that encodes aPPARα derived from a mammal heterologous to the non-human mammal (forexample, mouse) to be the subject of gene transfer (for example, human,bovine, monkey, swine, sheep, goat, rabbit, dog, cat, guinea pig,hamster, rat and the like), or a protein having substantially the sameamino acid sequence as the PPARα. Preferably, the DNA that encodes aheterologous PPARα of the present invention is a DNA that encodes thehuman PPARα or a protein having substantially the same amino acidsequence as the human PPARα. As the DNA that encodes the human PPARα, aDNA that encodes the amino acid sequence shown by SEQ ID NO: 2,preferably a DNA that has the base sequence shown by SEQ ID NO: 1, canbe mentioned. As examples of “substantially the same amino acidsequence”, in the case of the human PPARα, an amino acid sequencepossessing an identity of about 90% or higher, preferably 95% or higher,more preferably about 98% or higher, to the amino acid sequence shown bySEQ ID NO: 2, and the like can be mentioned. Amino acid sequenceidentity can be calculated using the homology calculation algorithm NCBIBLAST (National Center for Biotechnology Information Basic LocalAlignment Search Tool) under the following conditions (expectancy=10;gap allowed; matrix=BLOSUM62; filtering=OFF).

As examples of the “protein having substantially the same amino acidsequence”, in the case of the human PPARα, a protein havingsubstantially the same amino acid sequence as the amino acid sequenceshown by SEQ ID NO: 2, and possessing substantially the same quality ofactivity as a protein having the amino acid sequence shown by SEQ ID NO:2, is preferred. As examples of “substantially the same quality ofactivity”, ligand (including agonist and antagonist) binding activity,regulatory action on lipid metabolism-related gene expression and thelike can be mentioned. “Substantially the same quality of activity”indicates that their activities are qualitatively equivalent to eachother. Therefore, it is preferable that activities such asligand-binding activity and regulatory action on lipidmetabolism-related gene expression be equivalent (e.g., about 0.01-100folds, preferably about 0.5-20 folds, more preferably about 0.5-2folds), but quantitative factors such as the degrees of these activitiesand protein molecular weight may differ. Measurements of activities suchas ligand-binding activity and regulatory action on lipidmetabolism-related gene expression can be conducted in accordance with amethod known per se, and can be measured according to, for example, thescreening method described below.

Although the DNA that encodes a heterologous PPARα is preferably in anintron-free state (that is, a complementary DNA) like, for example, aDNA having the base sequence shown by SEQ ID NO: 1, an intron-containingform (that is, genomic DNA) can also be used preferably in another modeof embodiment because the 5′ and 3′ terminal sequences of the intron arecommon to almost all eukaryotic organism genes.

The DNA that encodes a heterologous PPARα can be isolated by thehybridization method or the PCR method and the like, with anoligonucleotide prepared on the basis of a known PPARα gene sequence asthe probe or primer, using a DNA derived from the liver, heart, kidney,adrenal gland, blood vessel, gastrointestinal tract and the like of ahuman or one of various non-human mammals (bovine, monkey, swine, sheep,goat, rabbit, dog, cat, guinea pig, hamster, rat, mouse and the like)and all or a portion of a genomic DNA derived from one of variouscommercially available genomic DNA libraries as the raw material, orusing a cDNA prepared from an RNA derived from the liver, heart, kidney,adrenal gland, blood vessel, gastrointestinal tract and the like of ahuman or one of various non-human mammals by a known method as the rawmaterial.

The transgenic animal of the present invention retains a DNA thatencodes a heterologous PPARα in “an expressible state”. Therefore, inintroducing the DNA to the subject animal, it is generally advantageousto use the DNA in a form containing an expression cassette joineddownstream of a promoter capable of functioning in the cells of thesubject animal (e.g., expression vector and the like).

As the vector that harbors a DNA that encodes a heterologous PPARα, anEscherichia coli-derived plasmid, a Bacillus subtilis-derived plasmid, ayeast-derived plasmid, a bacteriophage such as λ phage, a retrovirussuch as Molony leukemia virus, an animal or insect virus such asvaccinia virus or baculovirus, and the like are used. Particularly, anEscherichia coli-derived plasmid, a Bacillus subtilis-derived plasmid ora yeast-derived plasmid and the like are preferably used, and anEscherichia coli-derived plasmid is especially preferable.

As examples of the promoter for regulation of the gene expression of aheterologous PPARα, promoters of genes derived from viruses(cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virusand the like), promoters of genes derived from various mammals (human,bovine, monkey, swine, sheep, goat, rabbit, dog, cat, guinea pig,hamster, rat, mouse and the like) and birds (chicken and the like) [forexample, albumin, endothelin, osteocalcin, muscular creatine kinase,collagen type I and type II, cyclic AMP-dependent protein kinase βIsubunit, atrial natriuretic factor, dopamine β-hydroxygenase,neurofilament light chain, metallothionein I and IIA, metalloproteinase1 tissue inhibitor, smooth muscle α actin, polypeptide chain elongationfactor 1α (EF1-α), β actin, α and β myosin heavy chains, myosin lightchains 1 and 2, myelin basic protein, serum amyloid P component, reninand the like], and the like can be mentioned. Preferably, a promotercapable of expressing a heterologous PPARα specifically or at a highlevel in the target tissue according to the desired disease model (e.g.,gene promoters expressible at high levels in the liver, such as serumamyloid P component (SAP), albumin, transferrin, fibrinogen,antithrombin III, α1-antitrypsin; gene promoters expressible at highlevels in the heart, such as α and β myosin heavy chains and myosinlight chains 1 and 2; gene promoters expressible at high levels in thekidney, such as PTH/PTHrP receptor; gene promoters expressible at highlevels in the adrenal gland, such as ACTH receptor; gene promotersexpressible at high levels in the gastrointestinal tract, such as fattyacid-binding proteins; gene promoters expressible at high levels in thebrain, such as myelin basic protein and glial fibrillary acidic protein,and the like) can be appropriately selected. For example, when thetransgenic animal of the present invention is a model of hyperlipidemiaor arteriosclerosis, it is preferable to use a promoter expressible at ahigh level in the liver.

Downstream of the DNA that encodes a heterologous PPARα, a sequence thatterminates the transcription of the desired messenger RNA(polyadenylation (polyA) signal, also referred to as terminator) in thetransgenic animal is preferably present; for example, using a terminatorsequence derived from a virus gene, or derived from a gene of one ofvarious mammals or birds, it is possible to achieve efficient expressionof the transferred gene. Preferably, the simian virus SV40 terminatorand the like are used. In addition, to express the desired gene at astill higher level, the splicing signal of each gene, an enhancerregion, or a portion of the intron of an eukaryotic gene can be joinedupstream of the 5′ of the promoter region, between the promoter regionand the translational region, or downstream of the 3′ of thetranslational region, depending on the purpose.

Also, when a transgenic animal is prepared using an embryonic stem cell(ES cell), the above-described vector preferably further contains aselection marker gene for selection of a clone having the transferredDNA incorporated stably therein (e.g., drug resistance genes such as theneomycin resistance gene and the hygromycin resistance gene).Furthermore, when it is intended to incorporate the transferred DNA in aparticular site of a host chromosome by homologous recombination (thatis, preparation of a knock-in animal), the above-described vectorpreferably further contains, to eliminate random insertions, the herpessimplex virus-derived thymidine kinase (HSV-tk) gene or the diphtheriatoxin gene, as the negative selection marker gene, outside a DNAsequence homologous to the target site. These modes of embodiment aredescribed in detail below.

The above-described promoter, a DNA that encodes a heterologous PPARα, aterminator and the like can be inserted to the above-described vector inthe correct arrangement, that is, in an arrangement that enables theexpression of the heterologous PPARα in the transgenic animal, byordinary gene engineering techniques using appropriate restrictionenzyme and DNA ligase and the like.

In another preferred mode of embodiment, the expression vectorcontaining a DNA that encodes a heterologous PPARα obtained as describedabove, is transferred to an early embryo of the subject non-human mammalby the microinjection method.

An early embryo of the subject non-human mammal can be obtained bycollecting an internally fertilized egg obtained by mating a female anda male of the same species of non-human mammal, or by externallyfertilizing an ovum and sperm collected from a female and a male,respectively, of the same species of non-human mammal.

The age, breeding conditions and the like for the non-human mammal usedvary depending on the animal species, for example; when using the mouse(preferably an inbred mouse such as C57BL/6J (B6), F₁ between B6 andanother inbred line, and the like), it is preferable that the female beat about 4 to about 6 weeks of age, and the male be at about 2 to about8 months or so of age, and is also preferable that they be reared underthe conditions of about 12-hour light period (for example, 7:00-19:00)for about 1 week.

Although internal fertilization may be by spontaneous mating, a methodwherein for the purpose of regulating the sexual cycle and obtaining alarge number of early embryos from one animal, gonadotropine isadministered to a female non-human mammal to induce over-ovulation, andthereafter the female is mated with a male non-human mammal, ispreferred. As examples of the method of inducing ovulation in a femalenon-human mammal, a method wherein follicle-stimulating hormone(pregnant mare's serum gonadotropine, generally abbreviated as PMSG) isfirst administered, then luteinizing hormone (human chorionicgonadotropine, generally abbreviated as hCG) is administered, by, forexample, intraperitoneal injection and the like, is preferred; thepreferable hormone dosage and administration interval respectively varydepending on the species of non-human mammal. For example, when thenon-human mammal is the mouse (preferably an inbred mouse such asC57BL/6J (B6), F₁ between B6 and another inbred line, and the like), amethod wherein a fertilized egg is obtained by administering luteinizinghormone at about 48 hours after administration of follicle-stimulatinghormone, and thereafter immediately mating the female with a male mouse,is usually preferred; the dosage of follicle-stimulating hormone isabout 20-about 50 IU/animal, preferably about 30 IU/animal, and thedosage of luteinizing hormone is about 0-about 10 IU/animal, preferablyabout 5 IU/animal.

After a given time has elapsed, the abdominal cavity of each femalenon-human mammal confirmed by vaginal plug testing and the like to havecopulated was opened, and fertilized eggs are taken out from theoviduct, washed in a medium for embryo culture (e.g., M16 medium,modified Whitten medium, BWW medium, M2 medium, WM-HEPES medium,BWW-HEPES medium and the like) to remove cumulus cells, and cultured bythe droplet culture method and the like in the presence of 5% carbondioxide gas/95% atmosphere until the time of DNA microinjection. Whenmicroinjection is not immediately conducted, it is also possible topreserve the collected fertilized eggs under freezing by the slow methodor the ultrarapid method and the like.

On the other hand, in the case of external fertilization,follicle-stimulating hormone and luteinizing hormone are administered toa female non-human mammal for egg collection (the same as in the case ofinternal fertilization used preferably) in the same manner as above toinduce ovulation, after which eggs are collected and cultured in amedium for fertilization (e.g., TYH medium) until the time of externalfertilization by the droplet culture method and the like in the presenceof 5% carbon dioxide gas/95% atmosphere. On the other hand, the tail ofthe epididymis is taken out from the same species of male non-humanmammal (the same as in the case of internal fertilization is preferablyused), and a sperm mass is collected and pre-cultured in a medium forfertilization. After completion of the pre-culture, the sperm is addedto an egg-containing medium for fertilization; after cultivation by thedroplet culture method and the like in the presence of 5% carbon dioxidegas/95% atmosphere, fertilized eggs having two pronucleuses are selectedunder a microscope. When DNA microinjection is not immediatelyconducted, it is also possible to preserve the collected fertilized eggsunder freezing by the slow method or the ultrarapid method and the like.

DNA microinjection to a fertilized egg can be performed using a knownapparatus such as a micromanipulator according to a conventional method.Briefly speaking, the fertilized egg placed in a droplet of a medium forembryo culture is aspirated and immobilized using a holding pipette, anda DNA solution is injected directly to the male or female pronucleus,preferably into the male pronucleus, using an injection pipette. Thetransferred DNA used is preferably one that has been highly purified byCsCl density ultracentrifugation and the like. Also, the transferred DNAis preferably linearized by cutting the vector portion thereof using arestriction endonuclease.

After the DNA transfer, the fertilized egg is cultured in a medium forembryo culture by the droplet culture method and the like in thepresence of 5% carbon dioxide gas/95% atmosphere until the 1-cellstage—blastcyst stage, after which it is transplanted into the oviductor uterus of a female non-human mammal for embryo reception rendered tobe pseudopregnant. The female non-human mammal for embryo reception maybe any female, as long as it is of the same species as the animal fromwhich the early embryo to be transplanted is derived; for example, whena mouse early embryo is transplanted, a female ICR strain mouse(preferably about 8 to about 10 weeks of age) and the like arepreferably used. As an example of the method of rendering the femalenon-human mammal for embryo reception to be in a pseudopregnant state, amethod wherein the female is mated with the same species of vasectomized(vasoligated) male non-human mammal (for example, in the case of amouse, a male ICR strain mouse (preferably about 2 months or more ofage)), and selecting one confirmed as having a vaginal plug, is known.

The female for embryo reception used may be a spontaneously ovulatingfemale, or a female having fertility induced by administeringluteinizing hormone-releasing hormone (generally abbreviated as LHRH) oran analog thereof prior to mating with a vasectomized (vasoligated)male. As examples of the LHRH analog, [3,5-DiI-Tyr⁵]-LH-RH,[Gln⁸]-LH-RH, [D-Ala⁶]-LH-RH, [des-Gly¹⁰]-LH-RH, [D-His (Bzl)⁶]-LH-RH,Ethylamides thereof and the like can be mentioned. The dosage of LHRH oran analog thereof, and the timing of mating with a male non-human mammalafter administration thereof vary depending on the species of non-humanmammal. For example, when the non-human mammal is the mouse (preferablyan ICR strain mouse and the like), it is usually preferable that thefemale mouse be mated with a male mouse at about 4 days after LHRH or ananalog thereof is administered; the dosage of LHRH or an analog thereofis usually about 10-60 μg/animal, preferably about 40 μg/animal.

Usually, when the early embryo to be transplanted is in the morula stageor after, it is transplanted to the uterus of a female for embryoreception; when the early embryo is in an earlier stage (for example,1-cell stage to 8-cell stage embryo), it is transplanted to the oviduct.As the female for embryo reception, one which is older than a givennumber of days from pseudopregnancy, depending on the developmentalstage of the transplanted embryo, is appropriately used. For example, inthe case of the mouse, a female mouse at about 0.5 days afterpseudopregnancy is preferred for transplantation of a 2-cell stageembryo, and a female mouse at about 2.5 days after pseudopregnancy ispreferred for transplantation of a blastcystic embryo. After the femalefor embryo reception is anesthetized (preferably Avertin, Nembutal andthe like are used), an incision is made, the ovary is drawn out, earlyembryo (about 5 to about 10 cells) in suspension in a medium for embryoculture are injected to the peritoneal orifice of the oviduct or thevicinity of the oviduct junction of the uterine horn using a pipette forembryo transplantation.

If the transplanted embryo successfully implants and the embryorecipient female becomes pregnant, non-human mammal pups are obtained byspontaneous delivery or caesarian section. Embryo recipient females thatdelivered spontaneously are allowed to continue suckling; if the pupsare delivered by caesarian section, the pups can be suckled by aseparately provided female for suckling (for example, in the case of themouse, a female mouse with usual mating and delivery (preferably femaleICR strain mouse and the like)).

Referring to the introduction of a DNA that encodes a heterologous PPARαin the fertilized egg cell stage, it is assured that the transferred DNAis present in all germ line cells and somatic cells of the subjectnon-human mammal. Whether or not the transferred DNA is incorporated inthe chromosome DNA can, for example, be determined by screeningchromosome DNAs separated and extracted from the tails of offspringpups, by Southern hybridization or PCR method. The presence of a DNAthat encodes a heterologous PPARα in the germ line cells of non-humanmammal pups (F₀) obtained as described above means that a DNA thatencodes a heterologous PPARα is present in all of the germ line cellsand somatic cells of all progeny (F₁) animals.

Usually, the F₀ animals are obtained as heterozygotes having thetransferred DNA in only one of the homologous chromosomes. Also,individual F₀ animals are randomly inserted onto different chromosomesunless produced by homologous recombination. To obtain a homozygotehaving a DNA that encodes a heterologous PPARα on both of the homologouschromosomes, an F₀ animal and a non-transgenic animal are crossed toprepare F₁ animals, and siblings of a heterozygote having thetransferred DNA in only one of the homologous chromosomes are crossed toeach other. Provided that the transferred DNA has been incorporated inonly one gene locus, ¼ of the obtained F₂ animals would be homozygotes.

In another preferred mode of embodiment, the expression vectorcontaining a DNA that encodes a heterologous PPARα is transferred to anES cell of the subject non-human mammal by a known method of geneintroduction such as electroporation method.

An ES cell refers to a cell which is derived from the inner cell mass(ICM) of a fertilized egg in the blastcyst stage, and which can becultured and maintained while retaining an undifferentiated state invitro. Cells in the ICM are cells that will form the embryo itself andare also stem cells on which all tissues, including germ cells, arebased. The ES cell may be of an already established cell line, and mayalso be newly established in accordance with the method of Evans andKaufman (Nature, Vol. 292, p. 154, 1981). For example, in the case of amouse ES cell, currently, an ES cell derived from the 129 strain mouseis generally used; however, since the immunological background isunclear, for example, an ES cell established from the C57BL/6 mouse orthe BDF₁ mouse (F₁ between C57BL/6 and DBA/2), which has been developedby improving the low number of eggs collectable from C57BL/6 by crossingwith DBA/2, and the like can also be used favorably for the purpose ofinstead obtaining an ES cell which is of a pure strain, and which has animmunologically clear genetic background and the like. Because the BDF₁mouse has the C57BL/6 mouse as the background, in addition to beingadvantageous in that the number of collectable eggs is large and theeggs are tough, ES cells derived therefrom are advantageously usable inthat the genetic background can be replaced with the C57BL/6 mouse bybeing back-crossed with the C57BL/6 mouse when the disease model mouseis prepared.

Preparation of an ES cell can, for example, be conducted as describedbelow. A blastcystic embryo is collected from the uterus of a post-matedfemale non-human mammal [when using a mouse (preferably an inbred mousesuch as C57BL/6J (B6), F₁ between B6 and another inbred line, and thelike), for example, a female mouse at about 8 to about 10 weeks or so ofage (about 3.5 days of gestation) mated with a male mouse at about 2months or more of age is preferably used] (or it is also possible tocollect an early embryo in the morula stage or before from the oviduct,and thereafter culture it in a medium for embryo culture in the samemanner as above until the blastcyst stage), and cultured on a layer ofappropriate feeder cells (for example, in the case of the mouse, aprimary fibroblast prepared from a mouse fetus, a known STO fibroblastline and the like), whereby some cells of the blastcyst aggregate toform an ICM which will differentiate into an embryo. This inner cellmass is trypsinized to dissociate the single cells, and dissociation andpassage are repeated while maintaining an appropriate cell density andconducting medium exchanges, whereby an ES cell is obtained.

Although the ES cell may be of either sex, a male ES cell is usuallymore convenient for preparation of a germ line chimera. Also, it isdesirable, also for saving labor for painstaking cultivation, that sexidentification be conducted as soon as possible. As an example of the EScell sex identification method, a method wherein the gene in the sexdetermination region on the Y chromosome is amplified and detected bythe PCR method can be mentioned. Using this method, the number of EScells can be reduced to about 1 colony (about 50 cells), in contrast tothe conventional practice that requires a cell number of about 10⁶ cellsfor karyotype analysis, so that primary selection of ES cells in theinitial stage of cultivation can be conducted by sex identification,which in turn makes it possible to significantly save labor in theinitial stage of cultivation because early selection of male cells hasbeen made possible.

Also, secondary selection can be conducted by, for example, confirmationof the chromosome number by the G-banding method, and the like. Althoughthe chromosome number of the ES cell obtained is desirably 100% of thenormal number, it is desirable that if the 100% level is difficult toachieve for the reasons of physical operation and the like at the timeof cell line establishment, gene transfer to the ES cell be followed byre-cloning into a normal cell (for example, in the case of the mouse, acell having a chromosome number of 2n=40).

The ES cell line thus obtained need to be carefully subcultured tomaintain the properties of undifferentiated stem cells. For example, amethod wherein the ES cell line is cultured on appropriate feeder cellslike the STO fibroblast, in the presence of LIF (1-10,000 U/ml), whichis known as a differentiation suppression factor, in a carbon dioxidegas incubator (preferably 5% carbon dioxide gas/95% air or 5% oxygen/5%carbon dioxide gas/90% air) at about 37° C., and the like, and forpassage, for example, the ES cell line is rendered to be single cells bya treatment with a trypsin/EDTA solution (usually 0.001 to 0.5%trypsin/0.1 to 5 mM EDTA, preferably about 0.1% trypsin/1 mM EDTA), andsown onto freshly provided feeder cells, and the like are used. Thispassage is usually conducted every 1-3 days, during which period thecells are examined; if a morphologically abnormal cell is found, thecultured cells are desirably discarded.

ES cells can be differentiated into various types of cells such asparietal muscle, visceral muscle, and myocardium by subjecting them tomonolayer culture until a high density is reached, or to suspensionculture until a cell aggregate is formed, under appropriate conditions[M. J. Evans and M. H. Kaufman, Nature, Vol. 292, page 154, 1981; G. R.Martin, Proc. Natl. Acad. Sci. U.S.A., Vol. 78, page 7634, 1981; T. C.Doetschman et al., Journal of Embryology and Experimental Morphology,Vol. 87, page 27, 1985]; the heterologous PPARα-expressing non-humanmammal cell of the present invention, obtained by differentiating an EScell having a DNA that encodes a heterologous PPARα transferred thereto,is useful in cell biological investigations of a heterologous PPARα invitro.

For gene transfer to an ES cell, any of the calcium phosphateco-precipitation method, electroporation method, lipofection method,retrovirus infection method, aggregation method, microinjection method,gene gun (particle gun) method, DEAE-dextran method and the like can beused; however, the electroporation method is generally chosen for thereasons of the capability of treating a large number of cellsconveniently and the like. For electroporation, ordinary conditions usedfor gene introduction to animal cells can be used as is; for example,electroporation can be conducted by trypsinizing ES cells in thelogarithmic growth phase to disperse them to obtain a dispersion ofsingle cells, suspending the dispersion in a medium to obtain a celldensity of 10⁶ to 10⁸ cells/ml, transferring the suspension to acuvette, adding 10 to 100 μg of a vector containing a DNA that encodesthe heterologous PPARα, and applying electric pulses of 200 to 600 V/cm.

Although the ES cell incorporating the transferred DNA can also betested by screening chromosome DNAs separated and extracted from acolony obtained by culturing a single cell on feeder cells, by Southernhybridization or PCR method, the greatest advantage of a transgenicsystem using an ES cell resides in that a transformant can be selectedat the cell stage with the expression of a drug resistance gene or areporter gene as the index. Therefore, the introduced vector used heredesirably further contains, in addition to an expression cassettecontaining a DNA that encodes a heterologous PPARα, a selection markergene such as a drug resistance gene (e.g., neomycin phosphotransferaseII (nptII) gene, hygromycin phosphotransferase (hpt) gene and the like)or a reporter gene (e.g., β-galactosidase (lacZ) gene, chloramphenicolacetyltransferase (cat) gene and the like). For example, when using avector containing the nptII gene as the selection marker gene, the EScell after gene transfer treatment is cultured in a medium containing aneomycin-series antibiotic, such as G418, each of the emerging resistantcolonies is transferred to a culture plate; after trypsinization andmedium exchanges are repeated, a portion thereof is reserved forcultivation, whereas the remainder is subjected to PCR or Southernhybridization to confirm the presence of the transferred DNA.

When an ES cell confirmed to have the transferred DNA incorporatedtherein is returned into an embryo derived from the same species ofnon-human mammal, it is incorporated in the ICM of the host embryo and achimeric embryo is formed. By transplanting this to a foster mother (afemale for embryo reception), and allowing development to continue, achimeric transgenic animal is obtained. If the ES cell has contributedto the formation of primordial germ cells, which will differentiate intoeggs and sperm in the chimeric animal, a germ line chimera would beobtained; by mating this, a transgenic non-human mammal having thetransferred DNA fixed genetically therein can be prepared.

As the method of preparing a chimeric embryo, there are a method whereinearly embryos up to the morula stage are adhered together and aggregated(aggregation chimera method) and a method wherein a cell ismicro-injected into a cleavage cavity of the blastcyst (injectionchimera method). Although the latter has traditionally been widelyconducted in the preparation of a chimeric embryo using an ES cell, amethod wherein an aggregate chimera is created by injecting an ES cellinto the zona pellucida of an 8-cell stage embryo, and a method whereinan aggregate chimera is created by co-culturing and aggregating an EScell mass and an 8-cell stage embryo with the zona pellucida removedtherefrom as a method which does not require a micromanipulator andwhich can be easily operated, have recently also been conducted.

In all cases, a host embryo can be collected in the same manner from anon-human mammal that can be used as the female for egg collection ingene transfer to a fertilized egg; for example, in the case of themouse, to enable a determination of the percent contribution of the EScell to chimeric mouse formation by fur color (coat color), it ispreferable to collect a host embryo from a mouse of a strain whose furcolor is different from that of the strain from which the ES cell isderived. For example, provided that the ES cell is derived from the 129strain mouse (fur color: agouti), the C57BL/6 mouse (fur color: black)or the ICR mouse (fur color: albino) can be used as the female for eggcollection; provided that the ES cell is derived from the C57BL/6 orDBF₁ mouse (fur color: black) or from the TT2 cell (F₁ between C57BL/6and CBA (fur color: agouti)), the ICR mouse or the BALB/c mouse (furcolor: albino) can be used as the female for egg collection.

Also, because the potential of germ line chimera formation dependslargely on the combination of ES cell and host embryo, it is morepreferable to select a combination showing a high germ line chimeraformation potential. For example, in the case of the mouse, it ispreferable to use a host embryo and the like derived from the C57BL/6strain for ES cells derived from the 129 strain, and host embryo and thelike derived from the BALB/c strain are preferred for ES cells derivedfrom the C57BL/6 strain.

The female mice for egg collection are preferably about 4 to about 6weeks or so of age; as the male mouse for mating, one of the same strainat about 2 to about 8 months or so of age is preferred. Although matingmay be by spontaneous mating, it is preferably conducted aftergonadotropic hormones (follicle-stimulating hormone, then luteinizinghormone) are administered to induce over-ovulation.

In the case of the blast disk injection method, a blastcystic embryo(for example, in the case of a mouse, at about 3.5 days after mating) iscollected from the uterus of a female for egg collection (or an earlyembryo in the morula stage or before, after being collected from theoviduct, may be cultured in the above-described medium for embryoculture until the blastcyst stage), and an ES cell having a DNA thatencodes a heterologous PPARα transferred thereto (about 10 to about 15cells) is injected into a cleavage cavity of the blastcyst using amicromanipulator, after which it is transplanted into the uterus of afemale non-human mammal for embryo reception rendered to bepseudopregnant. As the female non-human mammal for embryo reception, anon-human mammal usable as a female for embryo reception in genetransfer to a fertilized egg can be used in the same manner.

In the case of the co-culture method, an 8-cell stage embryo and morula(for example, in the case of the mouse, about 2.5 days after mating) arecollected from the oviduct and uterus of the female for egg collection(or it is also possible to collect an early embryo in the 8-cell stageor before from the oviduct, and thereafter culture it in theabove-described medium for embryo culture until the 8-cell stage or themorula stage); after the zona pellucida is dissolved in acidic Tyrode'ssolution, an ES cell mass having a DNA that encodes a heterologous PPARαtransferred thereto (number of cells: about 10 to about 15 cells) isplaced in a droplet of a medium for embryo culture layered with mineraloil, the above-described 8-cell stage embryo or morula (preferably 2cells) is further placed, and they are co-cultured overnight. Theobtained morula or blastcyst is transplanted into the uterus of a femalenon-human mammal for embryo reception in the same manner as above.

If the transplanted embryo successfully implants and the embryorecipient female becomes pregnant, chimeric non-human mammal pups areobtained by spontaneous delivery or caesarian section. Embryo recipientfemales that delivered spontaneously are allowed to continue suckling;if the female has delivered by caesarian section, the pups may besuckled by a separately provided female for suckling (a female non-humanmammal with usual mating and delivery).

For selection of a germ line chimera, first, a chimeric mouse, of thesame sex as the ES cell, provided that the sex of the ES cell isdetermined in advance, is selected (usually, a ale chimeric mouse isselected because a male ES cell is used), next, a chimeric mouse showinga high percent contribution of the ES cell, based on a phenotype such asfur color, is selected (for example, 50% or higher). For example, in thecase of a chimeric mouse obtained from a chimeric embryo of the D3 cell,which is a male ES cell derived from the 129 strain mouse, and a hostembryo derived from the C57BL/6 mouse, it is preferable to select a malemouse showing a high percentage of the agouti fur color. Whether or notthe selected chimeric non-human mammal is a germ line chimera can bedetermined on the basis of the phenotype of the F₁ animal obtained bycrossing with the same species of animal of the appropriate strain. Forexample, in the case of the above-described chimeric mouse, agouti isdominant over black; therefore, when the selected male mouse is crossedwith a female C57BL/6 mouse, the fur color of the obtained F₁ would beagouti, provided that the selected male is a germ line chimera.

The germ line chimeric non-human mammal having a DNA that encodes aheterologous PPARα transferred thereto (founder) thus obtained isusually obtained as a heterozygote having the transferred DNA only inonly one of the homologous chromosomes. Also, the individual foundersare randomly inserted onto different chromosomes unless based onhomologous recombination. To obtain a homozygote having a DNA thatencodes a heterologous PPARα on both of the homologous chromosomes, outof F₁ animals obtained as described above, heterozygous siblings havingthe transferred DNA only in one of the homologous chromosomes arecrossed to each other. Selection of heterozygotes can, for example, betested by screening chromosome DNA separated and extracted from the tailof the F₁ animal by Southern hybridization or PCR method. Provided thatthe transferred DNA has been incorporated in only one gene locus,one-fourth of the obtained F₂ animals would be homozygotes.

The transgenic animal of the present invention is not subject tolimitation as to expression of endogenous PPARα, as long as anexpression amount of heterologous PPARα is assured to the extent thatenables a quantitative-measurement of the action of the test substanceon a heterologous PPARα. However, when using a transgenic animal of thepresent invention to evaluate a drug that is capable of acting not onlyon a heterologous PPARα but also on an endogenous PPARα, it is desirablethat the expression of the endogenous PPARα be inactivated. A transgenicanimal of the present invention wherein the expression of endogenousPPARα has been inactivated can be obtained by introducing a DNA thatencodes the heterologous PPARα, according to the method described above,to an ES cell having the PPARα gene knocked out, selected by a knownmethod (see, for example, Lee S. S. et al., Molecular and CellularBiology (Mol. Cell. Biol.), Vol. 15, page 3012, 1995), or an earlyembryo or ES cell derived from a PPARα knock-out animal prepared fromthe ES cell according to the method described above. As a specific meansof knocking out a PPARα gene, a method wherein the PPARα gene derivedfrom the subject non-human mammal is isolated according to aconventional method, and a DNA chain having a DNA sequence constructedso that the gene is eventually inactivated by, for example, insertinganother DNA fragment (for example, drug resistance genes such as theneomycin resistance gene and the hygromycin resistance gene, reportergenes such as lacZ (β-galactosidase gene) and cat (chloramphenicolacetyltransferase gene) and the like) to the exon portion thereof todestroy the function of the exon (in this case, incorporation of thetransferred DNA can be selected with drug resistance or reporter geneexpression as the index as described above), or by cleaving out all or aportion of the PPARα gene out using the Cre-loxP system or the Flp-frtsystem to delete the gene, or by inserting the stop codon into theprotein-coding region to disable the complete translation of theprotein, or by inserting a DNA sequence that terminates thetranscription of the gene into the transcription region (for example,polyA adduct signal and the like) to disable the complete synthesis ofthe messenger RNA, (hereinafter abbreviated as targeting vector), isincorporated by homologous recombination into the PPARα gene locus ofthe subject non-human mammal, can be preferably mentioned.

Usually, gene recombinations in a mammal are mostly non-homologous; thetransferred DNA is randomly inserted at an optionally chosen position onthe chromosome. Therefore, it is not possible to efficiently select onlythose clones targeted to the target endogenous PPARα gene by homologousrecombination by selection based on detection of drug resistance orreporter gene expression and the like; it is necessary to confirm theincorporation site by the Southern hybridization method or the PCRmethod for all selected clones. Hence, provided that, for example, theHSV-tk gene, which confers gancyclovir sensitivity, has been joinedoutside of a region homologous to the target sequence of the targetingvector, the cells having the vector inserted randomly thereto areincapable of growing in a gancyclovir-containing medium because theyhave the HSV-tk gene, whereas the cells that have been targeted to theendogenous PPARα gene locus by homologous recombination become resistantto gancyclovir and are selected because they do not have the HSV-tkgene. Alternatively, provided that the diphtheria toxin gene, forexample, is joined in place of the HSV-tk gene, the cells having thevector inserted randomly thereto die due to the toxin produced thereby,so that a homologous recombinant can also be selected in the absence ofa drug.

Also, the transgenic animal of the present invention having theexpression of an endogenous PPARα inactivated can also be obtained bymating the thus-prepared animal having the endogenous PPARα knocked outtherein and an animal of the same species having the above-describedheterologous PPARα transferred thereto. For example, by further matingfemale and male pups obtained by mating an endogenous PPARαhomo-deficient mouse and a human PPARα homo-transgenic (Tg) mouse(endogenous PPARα hetero-deficient-human PPARα hetero-Tg mouse), andselecting a mouse confirmed to have 2 copies of the human PPARα and onlythe deficient portion of the knocked-out mouse PPARα, it is possible toprepare an endogenous PPARα homo-deficient-human PPARα homo-Tg mouse.

Alternatively, the transgenic animal of the present invention having theexpression of an endogenous PPARα inactivated may be a knock-in animalwherein an endogenous PPARα gene is substituted by a DNA that encodes aheterologous PPARα by gene targeting using homologous recombination.

A knock-in animal can be prepared according to basically the sametechnique as that for a knock-out animal. Because the ORF of PPARα ispresent in exon 3 to exon 8, for example, it is acceptable to cleavethese regions of a PPARα gene derived from the subject non-human mammalusing an appropriate restriction endonuclease, insert the correspondingregions of a heterologous PPARα gene instead to obtain a DNA-containingtargeting vector, introduce the vector to an ES cell derived from thesubject non-human mammal according to the above-described method, andselect an ES cell clone having a DNA that encodes the heterologous PPARαincorporated by homologous recombination to the endogenous PPARα genelocus of the animal. Although clone selection can be conducted using thePCR method or the Southern method, for example, a homologous recombinantcan be selected with drug resistance as an index, provided that a markergene for positive selection such as the neomycin resistance gene isinserted to the 3′ non-translational region and the like of the PPARαgene of the targeting vector, and a marker gene for negative selectionsuch as the HSV-tk gene or the diphtheria toxin gene is further insertedto the outside of a region homologous to the target sequence.

Also, because there are some cases in which the expression of theheterologous PPARα having a marker gene for positive selectiontransferred thereto is hampered, it is preferable that a targetingvector having the loxP sequence or the frt sequence arranged at bothends of a marker gene for positive selection be used, and the markergene for positive selection be cleaved out by allowing the Cre or Flprecombinase or an expression vector for the recombinase (e.g.,adenovirus vector and the like) to act at an appropriate time afterhomologous recombinant selection. Alternatively, it is also acceptableto arrange a sequence homologous to the target sequence at both ends ofa marker gene for positive selection repeatedly in the same direction,instead of using a Cre-loxP system or an Flp-frt system, and cleave outthe marker gene for positive selection by means of intragenicrecombination between the sequences.

The transgenic animal of the present invention is further characterizedin that, in addition to that a DNA that encodes a heterologous PPARα isstably retained in an expressible state, it has one or more differentgenetic modifications. The “different genetic modification” means agenetic modification other than introduction of a DNA that encodes aheterologous PPARα, and includes a spontaneously pathogenic diseasemodel animal having an endogenous gene modified by a spontaneousmutation, a transgenic animal having another gene further transferredthereto, a knock-out animal having an endogenous gene inactivated(including not only gene destructions due to an insertion mutation andthe like, but also transgenic animals wherein the expression of a genehas been decreased to the extent that is undetectable or negligible byintroduction of an antisense DNA or a DNA that encodes a neutralizingantibody), a dominant negative variant having a mutated endogenous genetransferred thereto, and the like. Therefore, a modification of anendogenous PPARα gene is also included in the scope of the “differentgenetic modification” in the present invention.

Although the “different genetic modification” is not subject tolimitation, as long as it is advantageous for the purpose of thetransgenic animal of the present invention for evaluation of thepharmacological effect of an agonist or antagonist specific for theheterologous PPARα; for example, the “different genetic modification” ispreferably a genetic modification that produces a pathological conditionequal or similar to the disease associated with the regulation of PPARαactivity.

“A disease wherein PPARα activity regulation is involved” should beunderstood as a concept including not only a disease which is caused byan abnormality of PPARα activity or which eventually produces anabnormality of PPARα activity, but also a disease for which aprophylactic and/or therapeutic effect can be obtained by regulatingPPARα activity. For example, as diseases that can be prevented/treatedby activating a PPARα, hyperlipidemia, hypertriglyceride-(TG)-mia,combined dyslipidemia, hypo-HDL-cholesterolemia, arteriosclerosis,peripheral arterial obstruction, intermittent claudication, gangrene,hypertension, thrombosis, ischemic heart disease, acute cardiacinfarction, cardiac failure, congestive cardiac failure, unstable anginapectoris, restenosis after PTCA (percutaneous transluminal coronaryangioplasty), post-stenting restenosis, hyperfibrinogemia, cardiacmyopathy, cerebral hemorrhage, transient cerebral ischemic stroke,cerebral infarction, stroke, chronic glomerulonephritis, diabeticnephropathy, renal arteriosclerosis, dermatitis, immunodeficiency,hypoglycemia, hypoketonemia, fatty liver, diabetes, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia,gonadal dysfunction and the like can be mentioned; as diseases that canbe prevented/treated by inhibiting a PPARα, liver cancer, breast cancerand endometritis and the like can be mentioned.

As examples of the “disease model with one or more different geneticmodifications that produce a pathological condition equal or similar tothe disease associated with the regulation of PPARα activity”, WHHLrabbit (having a mutation in the low-density lipoprotein receptor(LDLR); Watanabe Y., Atherosclerosis, Vol. 36, page 261, 1980), SHLM (aspontaneously pathogenic mouse with an apoE deletion mutation;Matsushima Y. et al., Mammalian Genome (Mamm. Genome), Vol. 10, page352, 1999), LDLR knock-out mouse (Ishibashi S. et al., Journal ofClinical Investigation (J. Clin. Invest.), Vol. 92, page 883, 1993),apoE knock-out mouse (Piedrahita J. A. et al., Proceedings of theNational Academy of Sciences (Proc. Natl. Acad. Sci. USA), Vol. 89, page4471, 1992), human apoA, human apoB double transgenic mouse (Callow M.J. et al., Proceedings of the National Academy of Sciences, USA (Proc.Natl. Acad. Sci. USA), Vol. 91, page 2130, 1994) and the like ashyperlipidemia or arteriosclerosis models; CD55 CD59 double transgenicmouse (Cowan P. J. et al., Xenotransplantation, Vol. 5, pages 184-90,1998) and the like as ischemic heart disease models; interleukin 1transgenic mouse (Groves R. W. et al., Proc. Natl. Acad. Sci. USA, Vol.92, page 11874, 1995) and the like as dermatitis models; CD19 knock-outmouse (Spielman J. et al., Immunity, Vol. 3, page 39, 1995) and the likeas immunodeficiency models; SPC2 knock-out mouse (Furuta M. et al.,Proc. Natl. Acad. Sci. USA, Vol. 94, page 6646, 1997) and the like ashypoglycemia models; ob/ob mouse (Herberg L. and Coleman D. L.,Metabolism (Metabolism), Vol. 26, page 59, 1977), KK mouse (Nakamura M.and Yamada K., Diabetologia, Vol. 3, page 212, 1967), FLS mouse (Soga M.et al., Laboratory of Animal Science (Lab. Anim. Sci.), Vol. 49, page269, 1999) as fatty liver model; NOD mouse (Makino S. et al.,Experimental Animal (Exp. Anim.), Vol. 29, page 1, 1980), BB rat (CrisaL. et al., Diabetes Metabolism Review (Diabetes Metab. Rev.), Vol. 8,page 4, 1992), ob/ob mouse, db/db mouse (Hummel L. et al., Science, Vol.153, page 1127, 1966), KK mouse, GK rat (Goto Y. et al., Tohoku Journalof Experimental Medicine (Tohoku J. Exp. Med.), Vol. 119, page 85,1976), Zucker fatty rat (Zucker L. M. et al., Annual of the New YorkAcademy of Science (Ann. NY Acad. Sci.), Vol. 131, page 447, 1965),OLETF rat (Kawano K. et al., Diabetes, Vol. 41, page 1422, 1992) and thelike as diabetes models; ob/ob mouse, db/db mouse, KK mouse, Zuckerfatty rat, OLETF rat and the like as obesity models; mutant amyloidprecursor protein transgenic mouse and the like as Alzheimer's diseasemodels; beta SAD (beta S-Antilles-D Punjab)transgenic mouse (Trudel M.et al., EMBO J, Vol. 10, page 3157, 1991) and the like as anemic hypoxiamodels; Steroidogenic factor 1 knock-out mouse (Zhao L. et al.,Development, page 128, page 147, 2001) and the like as gonadaldysfunction models; p53 knock-out mouse (Kemp C. J., MolecularCarcinogenesis, Vol. 12, page 132, 1995) and the like as liver cancermodels; c-neu transgenic mouse (Rao G. N. et al., Breast Cancer ResTreat, Vol. 48, page 265, 1998) and the like as breast cancer models;and perforin Fas-ligand double knock-out mouse (Spielman J. et al., JImmunol., Vol. 161, page 7063, 1998) and the like as endometritismodels, are known.

These “disease models with different genetic modification” can, forexample, be purchased from The Jackson Laboratory, USA, and the like, orcan easily be prepared using a commonly known genetic modificationtechnology.

A transgenic animal of the present invention may have been subjected toa non-genetic treatment that enables preparation of the same or anotherdisease model, in addition to “one or more genetic modifications thatproduce a pathological condition equal or similar to the diseaseassociated with the regulation of PPARα activity. “A non-genetictreatment” means a treatment that does not produce a gene modificationin the subject non-human mammal. As examples of such treatments,high-fat diet loading treatment, sugar loading treatment, starvationtreatment, vascular ligation/reperfusion and the like can be mentioned.

PPARα is expressed at high levels in the mammalian liver, heart, kidney,adrenal gland, gastrointestinal tract, brain and the like, and isassociated with diseases in these organs. Therefore, a transgenic animalof the present invention is preferably one that specifically expresses aheterologous PPARα at 1 or 2 or more sites out of the liver, heart,kidney, adrenal gland, gastrointestinal tract and brain. Suchsite-specific expression can be achieved using a promoter capable ofallowing the heterologous PPARα to be expressed specifically or at ahigh level in the target tissue described above. For example, by usingthe serum amyloid P component (SAP) promoter, which enables high-levelexpression in the liver, it is possible to prepare a transgenic animalof the present invention that expresses heterologous PPARα with liverspecificity or at high levels in the liver.

Also, when expressing PPARα at a considerably high level, some non-humanmammals having a DNA that encodes a heterologous PPARα transferredthereto exhibit a phenotype that develops a disease such as livercancer, breast cancer or endometritis, without having any differentgenetic modification. Therefore, the present invention also provides anon-human mammal which has a DNA that encodes a heterologous PPARαtransferred thereto, and which develops one or more diseases selectedfrom the group consisting of liver cancer, breast cancer andendometritis.

As another preferred mode of the “different genetic modification” thatis advantageous for the use of the transgenic animal of the presentinvention for evaluation of the pharmacological effect of an agonist orantagonist specific for the heterologous PPARα, introduction of aforeign DNA under the control of a PPRE-containing promoter can bementioned. As described above, PPARα binds to the PPRE sequence presentupstream of the 5′ of the target gene in the presence of a ligand topromote the transcription of the gene (or to suppress the transcriptionin the case of some genes (e.g., apoC-III and the like)). Therefore, ina non-human mammal having a foreign DNA under the control of aPPRE-containing promoter transferred thereto, the agonist or antagonistactivity of a test substance can be evaluated by determining the effectof the substance on the expression of the DNA. Here, “a foreign DNA”means a DNA gene-transferred artificially from outside, and encompassesnot only heterologous DNA but also homogenic DNA. As the “foreign DNAunder the control of a PPRE-containing promoter”, genes derived from ahuman or another mammal harboring the PPRE sequence in the promoterregion thereof (genomic DNA), which serve as essential target genes ofPPARα, for example, genes such as CYP4A11, CYP7A1, PAI-1, ApoA-I,ApoA-II, ApoC-III and Acyl-CoA, can be mentioned. Alternatively, achimeric DNA wherein an appropriate reporter gene (e.g., β-galactosidasegene, luciferase gene, chloramphenicol acetyltransferase gene, alkalinephosphatase gene, peroxidase gene and the like) is joined downstream ofan optionally chosen promoter containing the PPRE sequence derived fromthese genes (functional in the cells of the subject non-human mammal) isalso preferred.

The method of transferring one or more different genetic modificationsthat produce a pathological condition equal or similar to the diseaseassociated with the regulation of PPARα activity, or a foreign DNA underthe control of a PPRE-containing promoter, to a non-human mammal havinga DNA that encodes a heterologous PPARα transferred thereto, is notsubject to limitation; for example, a method wherein a non-human mammalhaving a DNA that encodes a heterologous PPARα transferred thereto andthe same species of non-human mammal having one or more differentgenetic modifications that produce a pathological condition equal orsimilar to the disease associated with the regulation of PPARα activity,or a foreign DNA under the control of a PPRE-containing promoter, arecrossed; a method wherein a DNA that encodes a heterologous PPARα istransferred, by the above-described method, to an early embryo or EScell of a non-human mammal having one or more different geneticmodifications that produce a pathological condition equal or similar tothe disease associated with the regulation of PPARα activity, or aforeign DNA under the control of a PPRE-containing promoter, to obtain atransgenic animal; a method wherein one or more different geneticmodifications that produce a pathological condition equal or similar tothe disease associated with the regulation of PPARα activity, or aforeign DNA under the control of a PPRE-containing promoter, istransferred to an early embryo or ES cell of a non-human mammal having aDNA that encodes a heterologous PPARα transferred thereto, by theabove-described method, or by a knock-out technology; and the like canbe mentioned. Also, when one or more different genetic modifications areby introduction of a foreign gene or dominant mutation gene thatproduces a pathological condition equal or similar to the diseaseassociated with the regulation of PPARα activity, it is possible toobtain a transgenic animal by simultaneously or sequentiallytransferring the foreign gene and the like and a DNA that encodes aheterologous PPARα transgenic animal, to an early embryo or ES cell of awild type on-human mammal. When different genetic modification isintroduction of a foreign DNA under the control of a PPRE-containingpromoter as well, it is possible to obtain a transgenic animal bysimultaneously or sequentially introducing the foreign DNA and a DNAthat encodes a heterologous PPARα. Furthermore, when one or moredifferent genetic modifications that produce a pathological conditionequal or similar to the disease associated with the regulation of PPARαactivity are by destruction of an endogenous gene, it is acceptable todesign a NA that encodes a heterologous PPARα to make it capable oftargeting the endogenous gene to be destroyed, and to transfer it to anES cell of a wild type non-human mammal. In this case, as the targetingvector, the same as those mentioned as examples with respect to thepreparation of the above-described knock-in animal, except that theendogenous PPARα gene is replaced with the endogenous gene to bedestroyed, can be preferably used.

When crossing a non-human mammal having a DNA that encodes aheterologous PPARα and the same species of a disease model non-humanmammal having one or more different genetic modifications that produce apathological condition equal or similar to the disease associated withthe regulation of PPARα activity (or a foreign DNA under the control ofa PPRE-containing promoter) are crossed, it is desirable to crosshomozygotes with each other. For example, the F₁ obtained by crossing ahomozygote having a DNA that encodes a heterologous PPARα incorporatedin one gene locus thereof and an apoE homo-deficient hyperlipidemia(arteriosclerosis) model is hetero with respect to both genes.One-sixteenth of the F₂ animals obtained by sibling mating of such F₁animals are heterologous PPARα homo-transgenic-apoE homo-deficient.

Because “the transgenic non-human mammal of the present invention”obtained as described above expresses, in addition to an endogenousPPARα (or in place thereof), a heterologous PPARα, it enables anevaluation in vivo of the pharmacological effect of an agonist orantagonist specific for a heterologous PPARα which possesses agonist orantagonist activity against a heterologous PPARα, but which does notpossess activity against an endogenous PPARα. Therefore, the presentinvention provides a screening method for a heterologous PPARα agonistor antagonist, characterized in that a test substance is applied to thetransgenic non-human mammal of the present invention or a portion of thebody thereof, and the agonist or antagonist activity of the substanceagainst a heterologous PPARα is tested.

Here, “agonist activity” refers to a property for specifically bindingto a PPARα to shift the equilibrium state between the active form (thatis, a state wherein the agonist, as a heterodimer with RXR, is capableof binding to PPRE to activate the transcription of the target gene) andthe inactive form of the PPARα toward higher activity, with the degreethereof being not subject to limitation. Therefore, the scope, of “asubstance possessing agonist activity (agonist)” also encompassespartial agonists, as well as what is called full agonists. On the otherhand, “antagonist activity” refers to a property for antagonisticallybinding to the ligand-binding site of a PPARα, but having no or almostno influence on the equilibrium state between the active form and theinactive form, or a property for binding to an optionally chosen site ofa PPARα to shift the equilibrium state between the active form and theinactive form of the PPARα toward lower activity. Therefore, “asubstance possessing antagonist activity (antagonist)”, as used in thepresent specification is understood to be defined as a conceptencompassing both what is called neutral antagonist and inverse agonist.

Specifically, in the screening method of the present invention, a testsubstance is administered to the transgenic non-human mammal of thepresent invention. As the test substance, in addition to a knownsynthetic compound, peptide, protein, DNA library and the like, forexample, tissue extract, cell culture supernatant and the like of amammal (for example, mouse, rat, swine, bovine, sheep, monkey, human andthe like) are used. It is desirable to confirm in advance that the testsubstance acts specifically on the heterologous PPARα, by conducting abinding experiment in vitro with each of an endogenous PPARα andheterologous PPARα of the transgenic non-human mammal of the presentinvention to confirm binding only to the heterologous PPARα, or byconducting an expression assay in each of a non-human mammal-derivedcell and a heterologous mammal-derived cell, each having a reporter geneunder the control of PPRE introduced thereto, to confirm activation ofthe expression of the reporter gene only in the latter, and the like.Provided that the endogenous PPARα has been knocked out in thetransgenic non-human mammal of the present invention, this preliminaryscreening can be omitted.

The PPARα agonist/antagonist activity of the test substance can, forexample, be tested with fatty acid β oxidation activity or accompanyingchanges in blood TG concentration, apoC-III production or accompanyingchanges in blood TG concentration, apoA-I production or accompanyingchanges in blood HDL-C concentration, apoA-II production or accompanyingchanges in blood HDL-C concentration, and the like as indices. These canbe measured in accordance with a method known per se, for example, themethod described in Chung, B. H., Segrest, J. P., Ray, M. J., Brunzell,J. D., Hokanson, J. E., Krauss, R. M., Beaudrie, K., and Cone, J. T.,1986. Methods Enzymol. 128: 181-209. In the transgenic non-human mammalof the present invention, wherein “different genetic modification” is“introduction of a foreign DNA under the control of a PPRE-containingpromoter”, the PPARα agonist/antagonist activity of the test substancecan easily be measured by examining the expression of the foreign DNA.

The thus-selected heterologous PPARα agonist can be used as a safelow-toxicity prophylactic/therapeutic drug in the animal from which theheterologous PPARα is derived, for diseases such as hyperlipidemia,hypertriglyceridemia, combined dyslipidemia, hypo-HDL-cholesterolemia,arteriosclerosis, peripheral arterial obstruction, intermittentclaudication, gangrene, hypertension, thrombosis, ischemic heartdisease, acute myocardial infarction, heart failure, congestive heartfailure, unstable angina pectoris, post-PTCA restenosis, post-stentingrestenosis, hyperfibrinogemia, cardiomyopathy, cerebral hemorrhage,transient cerebral ischemic attack, cerebral infarction, cerebralapoplexy, chronic glomerulonephritis, diabetic nephropathy, renalarteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia andgonadal dysfunction.

The heterologous PPARα agonist can, for example, be used orally as atablet, capsule, elixir, microcapsule and the like, with a sugar coatingmade as necessary, or parenterally in the form of a sterile solutionwith water or another pharmaceutically acceptable solution, or aninjection such as a suspension. The agonist can be formulated into apreparation by being admixed in a unit dose form required for generallyrecognized preparation making, along with a physiologically acceptablecarrier, a flavoring agent, a filler, a vehicle, an antiseptic, astabilizer, a binder and the like. The active ingredient content inthese preparations is appropriately chosen in consideration of thedosage described below.

As examples of additives that can be admixed in tablets, capsules andthe like, binders like gelatin, corn starch, tragacanth, and acacia;fillers like crystalline cellulose; swelling agents like corn starch,gelatin, and alginic acid; lubricants like magnesium stearate;sweetening agents like sucrose, lactose or saccharin; flavoring agentslike peppermint, Gaultheria adenothrix oil or cherry; and the like areused. When the formulation unit form is a capsule, the above-describedtype of material can further contain a liquid carrier like an oil orfat. A sterile composition for injection can be formulated according toan ordinary preparation design such as dissolving or suspending anactive substance, a naturally produced vegetable oil such as sesame oilor coconut oil, and the like in a vehicle like water for injection. Asexamples of aqueous solutions for injection, physiological saline, anisotonic solution containing glucose or other auxiliary drugs (forexample, D-sorbitol, D-mannitol, sodium chloride and the like) and thelike can be mentioned, which may be used in combination with anappropriate solubilizer, for example, an alcohol (for example., ethanoland the like), a polyalcohol (for example, propylene glycol,polyethylene glycol and the like), a non-ionic surfactant (for example,polysorbate 80™, HCO-50 and the like), and the like. As examples of oilysolutions, sesame oil, soybean oil and the like can be mentioned, whichmay be used in combination with solubilizers benzyl benzoate, benzylalcohol and the like. Also, the heterologous PPARα agonist may beformulated with a buffering agent (for example, phosphate buffersolution, sodium acetate buffer solution and the like), a soothing agent(for example, benzalkonium chloride, procaine hydrochloride and thelike), a stabilizer (for example, human serum albumin, polyethyleneglycol and the like), a preservative (for example, benzyl alcohol,phenol and the like), an antioxidant and the like. The preparedinjection solution is usually filled in an appropriate ampoule.

Also, when the heterologous PPARα agonist is a nucleic acid such as aDNA or an RNA, the nucleic acid, alone, or after the DNA (or DNAcorresponding to the RNA) is inserted to an appropriate vector such asretrovirus vector, adenovirus vector, or adenovirus-associated virusvector, can be administered to a human or another mammal according to astandard means. The nucleic acid, as is, or after being formulated intoa preparation along with a physiologically acceptable carrier such as anauxiliary for promotion of ingestion, can be administered using a genegun or a catheter like a hydrogel catheter.

Because the preparation thus obtained is safe and of low toxicity, itcan be administered to, for example, a mammal having the same orsubstantially the same PPARα as the heterologous PPARα (for example,human, rat, mouse, guinea pig, rabbit, sheep, swine, bovine, horse, cat,dog, monkey and the like; “substantially the same” has the samedefinition as that given above), preferably an animal from which theheterologous PPARα is derived (preferably human).

The dosage of the heterologous PPARα agonist varies depending on thetarget disease, the subject of administration, the route ofadministration and the like; for example, when the agonist is orallyadministered for the purpose of treating hyper-TG-emia, the dosage in anadult (body weight 60 kg) is generally about 0.1 mg to 100 mg,preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg perday. In the case of parenteral administration, the dosage of the agonistvaries depending on the subject of administration, target disease andthe like; for example, when the agonist is administered for the purposeof treating hyper-TG-emia as an injection to an adult (body weight 60kg), the dosage is about 0.01 to 30 mg or so, preferably about 0.1 to 20mg or so, more preferably about 0.1 to 10 mg or so per day. When thesubject of administration is a non-human animal as well, a dosagecalculated per 60 kg body weight can be administered.

Also, a heterologous PPARα antagonist selected by the screening methodof the present invention can be used as a safe low-toxicityprophylactic/therapeutic drug in a mammal (preferably a mammal fromwhich the heterologous PPARα is derived, more preferably human), fordiseases such as liver cancer, breast cancer or endometritis. Theheterologous PPARα antagonist can be formulated into a preparation bythe same method as that for the above-described heterologous PPARαagonist, and can be orally or parenterally administered to a mammal (forexample, human, rat, mouse, guinea pig, rabbit, sheep, swine, bovine,horse, cat, dog, monkey and the like; “substantially the same” has thesame definition as that given above).

The dosage of the heterologous PPARα antagonist varies depending ontarget disease, the subject of administration, the route ofadministration and the like; for example, when the antagonist is orallyadministered for the purpose of treating liver cancer, the dosage in anadult (body weight 60 kg) is generally about 0.1 mg to 100 mg,preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg, perday. In the case of parenteral administration, the dosage of theantagonist varies depending on the subject of administration, targetdisease and the like; for example, when the antagonist is administeredfor the purpose of treating liver cancer as an injection to an adult(body weight 60 kg), the dosage is about 0.01 to 30 mg or so, preferablyabout 0.1 to 20 mg or so, more preferably about 0.1 to 10 mg or so, perday. When the subject of administration is a non-human animal as well, adosage calculated per 60 kg body weight can be administered.

Because “the transgenic non-human mammal of the present invention”produces a pathological condition equal or similar to the diseaseassociated with the regulation of PPARα activity, it is possible toscreen for a substance possessing prophylactic/therapeutic activityagainst a disease in a mammal (preferably human) from which theheterologous PPARα is derived, and in which the regulation of theactivity of the PPARα is involved, by administering a test substance tothe animal, and testing the effect of the substance on the pathologicalcondition. The pathological condition serving as an index can beappropriately selected depending on the kind of disease model; forexample, the prophylactic/therapeutic effect of the test substanceagainst disease can be evaluated with an improvement in bloodcholesterol concentration (total cholesterol concentration, TGconcentration, HDL-C concentration and the like) and the like as theindex when the transgenic non-human mammal of the present invention is ahyperlipidemia model, and with arteriosclerosis lesion involution andthe like as the index when the same is an arteriosclerosis model, bloodflow improvement and the like as the index when the same is a model ofischemic heart disease or cerebral vascular disease, and with cancerfocus involution and the like as the index when the same is a model ofliver cancer or breast cancer.

As described above, when expressing PPARα at a considerably high level,some non-human mammals having a DNA that encodes a heterologous PPARαtransferred thereto exhibit a phenotype that develops a disease such asliver cancer, breast cancer or endometritis, without having anydifferent genetic modification. Therefore, by administering the testsubstance to such a heterologous PPARα-transferred non-human mammal inthe same way, and testing the effect to ameliorate the pathologicalcondition of the above-described disease, substances possessingprophylactic/therapeutic activity for the disease can also be screenedfor.

The thus selected substance can be used as a safe low-toxicityprophylactic/therapeutic drug for diseases such as hyperlipidemia,hypertriglyceridemia, combined dyslipidemia, hypo-HDL-cholesterolemia,arteriosclerosis, peripheral arterial obstruction, intermittentclaudication, gangrene, hypertension, thrombosis, ischemic heartdisease, acute myocardial infarction, heart failure, congestive heartfailure, unstable angina pectoris, post-PTCA restenosis, post-stentingrestenosis, hyperfibrinogemia, cardiomyopathy, cerebral hemorrhage,transient cerebral ischemic attack, cerebral infarction, cerebralstroke, chronic glomerulonephritis, diabetic nephropathy, renalarteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes, diabetic neuropathy, diabeticretinopathy, obesity, Alzheimer's disease, anemic hypoxia, gonadaldysfunction, liver cancer, breast cancer and endometritis. the selectedsubstance can be formulated into a preparation by the same method asthat for the above-described heterologous PPARα agonist, and orally orparenterally administered to a mammal (for example, human, rat, mouse,guinea pig, rabbit, sheep, swine, bovine, horse, cat, dog, monkey andthe like).

The dosage of the selected substance varies depending on target disease,the subject of administration, the route of administration and the like;for example, when the substance is orally administered for the purposeof treating hyper-TG-emia, the dosage in an adult (body weight 60 kg) isgenerally about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, morepreferably about 1.0 to 20 mg per day. In the case of parenteraladministration, the dosage of the substance varies depending on thesubject of administration, target disease and the like; for example,when the substance is administered for the purpose of treatinghyper-TG-emia as an injection to an adult (body weight 60 kg), thedosage is about 0.01 to 30 mg or so, preferably about 0.1 to 20 mg orso, more preferably about 0.1 to 10 mg or so per day. When the subjectof administration is a non-human animal as well, a dosage calculated per60 kg body weight can be administered.

Sequence identification numbers in the sequence listing in the presentspecification indicate the following sequences.

[SEQ ID NO:1] Shows the base sequence of cDNA encoding human PPARa.

[SEQ ID NO:2] Shows the amino acid sequence of human PPARa.

[SEQ ID NO:3] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying human SAP promoter.

[SEQ ID NO:4] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying human SAP promoter.

[SEQ ID NO:5] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying rabbit β-globin enhancer.

[SEQ ID NO:6] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying rabbit β-globin enhancer.

[SEQ ID NO:7] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying cDNA fragment of human PPARa.

[SEQ ID NO:8] Shows the base sequence of an oligonucleotide designed tofunction as a primer for amplifying cDNA fragment of human PPARa.

[SEQ ID NO:9] Shows the base sequence of an oligonucleotide designed tofunction as a fluorescent probe to detect cDNA fragment of human PPARaamplified by PCR.

When bases, amino acids and the like are shown in abbreviations in thepresent specification, they are based on the abbreviations by theIUPAC-IUB Commission on Biochemical Nomenclature or abbreviationsconventionally used in the pertinent field; examples thereof are givenbelow.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediamine tetraacetate

SDS: Sodium dodecyl sulfate

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gln: Glutamine

pGlu: Pyroglutamic acid

Me: Methyl group

Et: Ethyl group

Bu: Butyl group

Ph: Phenyl group

TC: Thiazolyzine-4 (R)-carboxamide group

The present invention is described in more detail by means of thefollowing Examples, which are not to be construed as limiting the scopeof the present invention.

EXAMPLE 1 Construction of PPARα Vector

The hSAP (human serum amyloid P component) promoter, which is necessaryfor high-level expression of the introduced gene in the liver was clonedby PCR. From the human genome data in GENBANK, it is known that hSAP ispresent on chromosome 1. Since the 5′ upstream region could beidentified by search of homology with the cDNA sequence, the primers SA1(5′-ACTGAGTAGAAGTAGCAGAA-3′ (SEQ ID NO: 3)) and SA4(5′-CAGCGGCTTGTTCATATTCC-3′ (SEQ ID NO: 4)) were designed on the basisof the genome sequence of a promoter region used in the literature (J.Biol. Chem., 111, 736, 1992; Dev. Genet., 10, 336, 1989), a 0.67 KbpHindIII-AvrII fragment adjoining to the initiation codon was amplifiedby PCR (Takara Shuzo Ex Taq DNA polymerase used; treatment at 94° C. for2 minutes followed by treatment at 94° C. for 0.5 minutes, at 60° C. for0.5 minutes, and at 68° C. for 1 minute in 30 cycles), the obtainedfragment was cloned into the pCR2.1 vector (invitrogen Company) by theTA cloning (invitrogen Company) method, and the sequence was identified.Four clones were analyzed, and one clone lacking base substitution wasobtained (plasmid name: pCR2.1-SA4).

Next, cloning of a rabbit β-globin enhancer that is necessary forhigh-level expression in host mammalian cells was conducted by themethod described below. With genomic DNA prepared from rabbit blood asthe template, and using primers having a restriction endonuclease sitefor expression vector construction added to the 5′ terminus thereof,namely BG2 (5′-TCCTAGGTGAGAACTTCAGGGTGAGTTTG-3′ (SEQ ID NO: 5); with anAvrII site added) and BG3 (5′-CGGTACCTTTGCCAAAATGATGAGACAGC-3′ (SEQ IDNO: 6); with a KpnI site added), PCR (Takara Shuzo Ex Taq DNA polymeraseused; treatment at 94° C. for 2 minutes followed by treatment at 94° C.for 0.5 minutes, at 60° C. for 0.5 minutes, and at 72° C. for 1 minutein 30 cycles) was conducted to obtain an about 640 bp amplified enhancerregion. The amplified fragment was cloned into the pCR2.1-TOPO vector byTA cloning (invitrogen Company); 8 clones were examined for sequenceconfirmation (ABI Company BigDye Terminator used). As a result of acomparison of the sequence analysis results for the 8 clones withenhancer sequences, the sequences of the 8 clones generally agreed withknown sequences, so that they were judged to be derived from the rabbitgenome of the PCR template.

The PPARα cDNA used was a 1.4 Kbp KpnI-SalI fragment cleaved out frompMCMV-neo-hPPARα (Biochem. Biophys. Res. Commun., 278: 704-711 (2000)).For SV40 polyA, the fragment from BglII to HindIII containing the polyAadduct signal derived from pTB399 (R. Sasada et al., Cell Structure andFunction, 12: 205, 1987) was cloned into pSP73 (stratagene Company);after an SalI linker was added to the BglII site, the fragment was usedas a 0.27 Kbp SalI-HindIII fragment.

In constructing an expression vector, fist, an AvrII-KpnI fragmentrecovered from pCR2.1-enh5 was ligated to the AvrII-KpnI site ofpCR2.1-SA4 using a Takara ligation kit (Takara Shuzo) to yieldpCR2.1-SAPENH1 incorporating the promoter and the enhancer. On the otherhand, an about 1.4 Kbp hPPARα cDNA prepared from pMCMV-neo-hPPARα and anabout 0.27 Kbp SV40 polyA fragment (R. Sasada et al., Cell Structure andFunction, 12: 205, 1987) were ligated to the KpnI-HindIII site ofpBluescriptII KS-(stratagene) to yield pKS-PPAR polyA1 incorporating thecDNA and polyA. Subsequently, a NotI-KpnI fragment recovered frompCR2.1-SAPENH1 (about 1.3 Kbp) and a KpnI-NotI fragment recovered frompMCMV-neo-hPPARα (about 1.7 Kbp) were ligated to the NotI site ofpBluescriptII KS-(stratagene) to complete the construction of theexpression vector (plasmid name: pKS-SEPP2; a transformant obtained bycloning this plasmid into the Escherichia coli JM109 strain (Escherichiacoli JM109/pKS-SEPP2) has been deposited under accession number FERMBP-8151, since Aug. 14, 2002, at the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology at Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan (zipcode: 305-8566).). The injection fragment used to prepare a transgenicmouse was cleaved out by cleavage with NotI, and has a size of about 3.0Kbp (FIG. 1).

EXAMPLE 2 Preparation of Transgenic Mouse Having Human PPARα TransferredThereto

To female mice for egg collection (C57BL/6J strain, 9-13 weeks of age),5 IU PMSG was intraperitoneally administered; after the animals werereared in a breeding room with a 12 hours light period/12 hours darkperiod for 2 days, 5 IU hCG was intraperitoneally administered, and theywere mated with male mice (C57BL/6J strain, 15 to 20 weeks old).Separately, spontaneously estrual females for embryo reception mice (ICRstrain, 9 to 20 weeks old) were mated with vasoligated male mice (ICRstrain, 15 to 20 weeks old). On the following day, the abdominal cavityof each female mouse for egg collection with copulation confirmed byvaginal plug formation was opened and the oviduct was taken out; in M2medium, fertilized eggs were taken out using tweezers under astereoscopic microscope. A fertilized egg from which cumulus cells hadleft was aspirated using a pipette, placed in a drop of M16 mediumcovered with mineral oil, and cultured at 37° C. in the presence of 5%CO₂ for 2 hours until injection.

A fertilized egg was placed in a drop of M2 medium covered with mineraloil, and aspirated and immobilized using a holding pipette. Theinjection fragment solution (0.3 to 1.0 μg/ml) prepared in Example 1 wasaspirated into an injection pipette, the male pronucleus of thefertilized egg was pricked with the injection pipette under astereoscopic microscope, and the injection fragment was injected. Aftercompletion of the injection, the fertilized egg was placed in a drop ofM2 medium covered with mineral oil, and cultured at 37° C. in thepresence of 5% CO₂ until transplantation to a female for embryoreception.

After a female for embryo reception mouse confirmed by vaginal plugformation to be in a pseudopregnant state was anesthetized withNembutal, an incision was made in the posterior back, a fat mass waspicked and drawn out using tweezers, and immobilized using forceps. Theovarian sac was torn using tweezers under a stereoscopic microscope, and10 to 15 fertilized eggs per each oviduct were injected to the oviductopening using a transfer pipette. The ovary and the oviduct werereturned into the body and the incision was sutured, after which theanimal was continued to be reared in a breeding room with 12-hour lightperiod/12-hour dark period. At 21 days after embryo transplantation,mouse pups (F₀) were born. The transmission of the transferred DNA tothe mouse pups was confirmed by cutting out an about 1 cm portion of thetail using scissors, isolating DNA from the tissue extract by aconventional method, and conducting the PCR method.

The F₀ animals confirmed as having the transferred DNA transmittedthereto were mated with the C57BL/6J mouse at a stage when they becamecapable of reproduction, and pups (F₁) were obtained. The transmissionof the transferred DNA was confirmed in the same manner as above, and F₁animals having the transferred DNA (siblings) were mated to obtainanimals homozygous with respect to the transferred DNA.

EXAMPLE 3 Gene Expression Analysis of Transgenic Mouse

To examine the human PPARα expression level in the transgenic mouseobtained in Example 2, the liver was collected from the obtainedtransgenic mouse at 5 to 6 weeks old, and total RNA was extracted usingISOGEN (manufactured by Nippon Gene Co., Ltd.). Next, on the basis of atotal RNA purified using an RNase-free DNase set (manufactured by QIAGENK.K.) and the RNeasy Mini Kit (manufactured by QIAGEN K.K.), a cDNA wasprepared by a reverse transcription reaction using the kit TaqManTranscription Reagents (manufactured by Applied Biosystems Company). Asthe primer for real-time PCR (TaqMan PCR), 5′-CGCCAGCACGGACGA-3′ (SEQ IDNO: 7) and 5′-TTGTCCCCACATATTCGACACTC-3′ (SEQ ID NO: 8) were used, andas the FAM (fluorescent 5-carboxyfluorescein)-labeled TaqMan probe,5′-CCCCCGGCAGTGCCCTGAA-3′ (SEQ ID NO: 9) was used. Furthermore, usingthe TaqMan PCR Master Mix (manufactured by Applied Biosystems Company),a Real-time PCR REACTION was conducted in a dedicated plate with 20 μlof each cDNA sample as the template. The reaction was conducted usingthe PCR apparatus 7700 sequence detector (manufactured by AppliedBiosystems Company) by a treatment at 50° C. for 2 minutes and at 95° C.for 10 minutes followed by a treatment at 95° C. for 15 seconds and at60° C. for 1 minute in 40 cycles. After the reaction, data analysis wasconducted according to the analytical manual. As a standard for thequantitation described above, expression of the GAPDH gene wasquantified using the same cDNA sample, and using the TaqMan Rodent GAPDHControl Reagent (manufactured by Applied Biosystems Company), and theresults were used for data value correction. As a result of theexperiment, the expression of human PPARα was confirmed in the liver ofthe transgenic mouse.

EXAMPLE 4 Mating with PPARα Homo-Deficient Mouse

The human PPARα homo-Tg mouse prepared in Example 2 and a PPARαhomo-deficient mouse (The Jackson Laboratory, USA) are mated to obtainpups. DNA is extracted from the tail of each mouse pup and subjected toSouthern blotting analysis; a parent mouse that delivered pups in all ofwhich the human PPARα and the knocked-out deficient portion of the mousePPARα gene were detected, is selected as a human PPARα hetero-Tg-mousePPARα hetero-deficient mouse. Next, the female and the male are mated.Gene identification is conducted by the same method; a mouse confirmedto have 2 copies of the human PPARα and only the deficient portion ofthe knocked-out mouse PPARα gene is selected as a human PPARαhomo-Tg-mouse PPARα homo-deficient mouse.

EXAMPLE 5 Mating with Various Hyperlipidemia (Arteriosclerosis) ModelMice

(1) Mating with apoE Homo-Deficient Mouse

Female and male pups obtained by mating the human PPARα homo-Tg mouseprepared in Example 2 and an apoE homo-deficient mouse that is a modelof hyperlipemic (arteriosclerotic) model (The Jackson Laboratory, USA;having C57BL/6J as the genetic background) are mated. DNA is extractedfrom the tail of each obtained mouse pup in the same manner as above,and subjected to Southern blotting analysis; DNA is extracted from thetail of each obtained mouse pup and subjected to Southern blottinganalysis; a parent mouse that delivered pups in all of which the DNA ofthe human PPARα and the knocked-out apoE gene were detected, is selectedas a human PPARα hetero-Tg-apoE hetero-deficient mouse. Next, the femaleand the male are mated. Gene identification is conducted by the samemethod; a mouse confirmed to have 2 copies of the human PPARα and onlythe deficient portion of the knocked-out apoE gene is selected as ahuman PPARα homo-Tg-apoE homo-deficient mouse.

(2) Mating with LDL Receptor Homo-Deficient Mouse

The human PPARα homo-Tg mouse prepared in Example 2 and an LDL receptorhomo-deficient mouse that is a hyperlipidemic (arteriosclerotic) diseasemodel [a mouse prepared by Takeda Chemical Industries, Ltd. (describedin Japanese Laid-Open Patent Publication No. 10-56915), or a mousestored by The Jackson Laboratory, USA; both have C57BL/6J as the geneticbackground] are mated to obtain pups. DNA is extracted from the tail ofeach obtained mouse pup in the same manner as above and subjected toSouthern blotting analysis; a parent mouse that delivered pups in all ofwhich the DNA of the human PPARα and the knocked-out LDL receptor genewere detected, is selected as a human PPARα hetero-Tg-LDL receptorhetero-deficient mouse. Next, the female and the male are mated. Geneidentification is conducted by the same method; a mouse confirmed tohave 2 copies of the human PPARα and only the deficient portion of theknocked-out LDL receptor gene is selected as a human PPARα homo-Tg-LDLreceptor homo-deficient mouse.

(3) Mating with Human apoA-I Homo-Tg Mouse

Females and males of pups obtained by mating the human PPARα homo-Tgmouse prepared in Example 2 and a human apoA-I homo-Tg mouse that is amodel of hyperlipemic (arteriosclerotic) model (The Jackson Laboratory,USA; having C57BL/6J as the genetic background) are mated. DNA isextracted from the tail of each obtained mouse pup in the same manner asabove and subjected to Southern blotting analysis; a parent mouse thatdelivered pups in all of which the DNA of the human PPARα and the humanapoA-I gene were detected, is selected as a human PPARα-human apoA-Ihetero-Tg mouse. Next, the female and the male are mated. Geneidentification of the obtained pups is conducted by the same method; amouse confirmed to have 2 copies of each of the human PPARα and humanapoA-I is selected as a human PPARα-human apoA-I homo-Tg mouse.

(4) Furthermore, in each of (1) to (3) above, the human PPARαhomo-Tg-mouse PPARα homo-deficient mouse prepared in Example 4, in placeof the human PPARα homo-Tg mouse prepared in Example 2, is mated witheach of the apoE homo-deficient mouse, the LDL receptor homo-deficientmouse and the human apoA-I Tg mouse through the same procedures.

INDUSTRIAL APPLICABILITY

The heterologous PPARα-transferred disease model non-human mammal of thepresent invention provides a screening system useful in the developmentof prophylactic/therapeutic drugs for various diseases wherein PPARαactivity regulation is involved as it enables an evaluation of the invivo pharmacological effect of a PPARα agonist or antagonist that actson the heterologous PPARα but does not act on endogenous PPARα of thenon-human mammal.

Free Texts in Sequence Listing

SEQ ID NO:3

shows an oligonucleotide designed to function as a primer for amplifyinghuman SAP promoter.

SEQ ID NO:4

shows an oligonucleotide designed to function as a primer for amplifyinghuman SAP promoter.

SEQ ID NO:5

shows an oligonucleotide designed to function as a primer for amplifyingrabbit β-globin enhancer.

SEQ ID NO:6

shows an oligonucleotide designed to function as a primer for amplifyingrabbit β-globin enhancer.

SEQ ID NO:7

shows an oligonucleotide designed to function as a primer for amplifyingcDNA fragment of human PPARα.

SEQ ID NO:8

shows an oligonucleotide designed to function as a primer for amplifyingcDNA fragment of human PPARα.

SEQ ID NO:9

shows an oligonucleotide designed to function as a fluorescent probe todetect cDNA fragment of human PPARα amplified by PCR.

1. A non-human mammal, which stably retains a DNA encoding aheterologous PPARα in an expressible state and has one or more differentgenetic modifications, or a part of its living body.
 2. The animal orthe part of its living body of claim 1, wherein at least one of saiddifferent genetic modifications results in pathological condition(s)equal or similar to disease(s) associated with the regulation of PPARαactivity.
 3. The animal or the part of its living body of claim 1,wherein at least one of said different genetic modifications isintroduction of a foreign DNA under the control of a promoter havingPPRE.
 4. The animal or the part of its living body of claim 1, whereinsaid heterologous PPARα is human derived PPARα.
 5. The animal or thepart of its living body of claim 1, wherein said heterologous PPARα hasthe same or substantially the same amino acid sequence as the amino acidsequence represented by SEQ ID NO:
 2. 6. The animal or the part of itsliving body of claim 1, wherein said non-human mammal is rabbit, dog,cat, guinea pig, hamster, mouse or rat.
 7. The animal or the part of itsliving body of claim 1, wherein said non-human mammal is mouse.
 8. Theanimal or the part of its living body of claim 1, herein said animalexpresses said heterologous PPARα in place of lacking its endogenousPPARα.
 9. The animal or the part of its living body of claim 8, whereinsaid animal is obtainable by crossing an endogenous PPARα-deficientanimal and the same species of animal that expresses a heterologousPPARα.
 10. The animal or the part of its living body of claim 8, whereinsaid endogenous PPARα is mouse-derived PPARα and said heterologous PPARαis human-derived PPARα.
 11. The animal or the part of its living body ofclaim 2, wherein said diseases associated with the regulation of PPARαactivity are one or more diseases selected from the group consisting ofhyperlipidemia, hypertriglyceridemia, combined dyslipidemia,hypo-HDL-cholesterolemia, arteriosclerosis, peripheral arterialobstruction, intermittent claudication, gangrene, hypertension,thrombosis, ischemic heart disease, acute myocardial infarction, heartfailure, congestive heart failure, unstable angina pectoris, post-PTCArestenosis, post-stenting restenosis, hyperfibrinogemia, cardiomyopathy,cerebral hemorrhage, transient ischemic attack, cerebral infarction,cerebral apoplexy, chronic glomerulonephritis, diabetic nephropathy,renal arteriosclerosis, dermatitis, immunodeficiency, hypoglycemia,hypoketonemia, fatty liver, diabetes mellitus, diabetic neuropathy,diabetic retinopathy, obesity, Alzheimer's disease, anemic hypoxia,gonadal dysfunction, liver cancer, breast cancer and endometritis. 12.The animal or the part of its living body of claim 1, wherein saidheterologous PPARα is specifically expressed in one or more regionselected from the group consisting of liver, heart, kidney, adrenalgland, blood vessel, gastrointestinal tract and brain.
 13. The animal orthe part of its living body of claim 1, wherein said heterologous PPARαis specifically expressed in liver.
 14. A method of screening for anagonist or antagonist for a heterologous PPARα, which comprises applyinga test substance to the animal or the part of its living body of claim1, and assaying its agonistic or antagonistic activity against theheterologous PPARα.
 15. A method of screening for an agonist orantagonist for a heterologous PPARα, which comprises applying a testsubstance to the animal or the part of its living body of claim 3, andassaying its agonistic or antagonistic activity against the heterologousPPARα using the expression of a foreign DNA under the control of apromoter having PPRE as an index.
 16. A method of screening for asubstance having a prophylactic/therapeutic activity for disease(s)associated with the regulation of PPARα activity in an animal from whicha heterologous PPARα is derived, which comprises administering a testsubstance to the animal of claim 2, and assaying effect(s) of thesubstance on pathological condition(s) equal or similar to disease(s)associated with the regulation of PPARα activity in the animal.